Ion Mobility Mass Spectrometry Research Articles

Full Native timsTOF PASEF-Enabled Quantitative Proteomics with the i2MassChroQ Software Package.

Ion mobility mass spectrometry has become popular in proteomics lately, in particular because the Bruker timsTOF instruments have found significant adoption in proteomics facilities. The Bruker's implementation of the ion mobility dimension generates massive amounts of mass spectrometric data that require carefully designed software both to extract meaningful information and to perform processing tasks at reasonable speed. In a historical move, the Bruker company decided to harness the skills of the scientific software development community by releasing to the public the timsTOF data file format specification. As a proteomics facility that has been developing Free Open Source Software (FOSS) solutions since decades, we took advantage of this opportunity to implement the very first FOSS proteomics complete solution to natively read the timsTOF data, low-level process them, and explore them in an integrated quantitative proteomics software environment. We dubbed our software i2MassChroQ because it implements a (peptide)identification-(protein)inference-mass-chromatogram-quantification processing workflow. The software benchmarking results reported in this paper show that i2MassChroQ performed better than competing software on two critical characteristics: (1) feature extraction capability and (2) protein quantitative dynamic range. Altogether, i2MassChroQ yielded better quantified protein numbers, both in a technical replicate MS runs setting and in a differential protein abundance analysis setting.

Quantitative Online Monitoring of an Immobilized Enzymatic Network by Ion Mobility-Mass Spectrometry.

The forward design of in vitro enzymatic reaction networks (ERNs) requires a detailed analysis of network kinetics and potentially hidden interactions between the substrates and enzymes. Although flow chemistry allows for a systematic exploration of how the networks adapt to continuously changing conditions, the analysis of the reaction products is often a bottleneck. Here, we report on the interface between a continuous stirred-tank reactor, in which an immobilized enzymatic network made of 12 enzymes is compartmentalized, and an ion mobility-mass spectrometer. Feeding uniformly 13C-labeled inputs to the enzymatic network generates all isotopically labeled reaction intermediates and products, which are individually detected by ion mobility-mass spectrometry (IMS-MS) based on their mass-to-charge ratios and inverse ion mobilities. The metabolic flux can be continuously and quantitatively monitored by diluting the ERN output with nonlabeled standards of known concentrations. The real-time quantitative data obtained by IMS-MS are then harnessed to train a model of network kinetics, which proves sufficiently predictive to control the ERN output after a single optimally designed experiment. The high resolution of the time-course data provided by this approach is an important stepping stone to design and control sizable and intricate ERNs.

Ion Mobility-Mass Spectrometry Strategies to Elucidate the Anhydrous Structure of Noncovalent Guest/Host Complexes.

Structural mass spectrometry (MS) techniques are fast and sensitive analytical methods to identify noncovalent guest/host complexation phenomena for desirable solution-phase properties. Current MS-based studies on guest/host complexes of drug and drug-like molecules are sparse, and there is limited guidance on how to interpret MS information in the context of host nanoencapsulation and inclusion. Here, we use structural MS strategies, combining energy-resolved MS (ERMS), ion mobility-MS (IM-MS), and computational modeling, to characterize 14 chemically distinct drug and drug-like compounds for their propensity to form guest/host complexes with the widely used excipient, beta-cyclodextrin (βCD). The majority (11/14) yielded a 1:1 guest/host complex, and ion mobility collision cross section (CCS) analysis provided subtle evidence of gas-phase compaction of complexes in both polarities. The three distinct dissociation channels observed in ERMS (i.e., charged βCD, charged guest, and partial guest loss) were used to direct charge-site assignments for computational modeling, and structural candidates were prioritized using helium-derived CCS measurements combined with root-mean-square distance analysis. The combined analytical information from ERMS, IM-MS, and computational modeling suggested that the majority of anhydrous complexes are inclusion complexes with βCD. Taken together, this work demonstrates a roadmap for how multiple MS-based analytical measurements can be combined to interpret the structures that guest/host complexes adopt in the absence of water.

Accurate Collisional Cross Section Measurement by Multipass Cyclic Ion Mobility Spectrometry.

Ion mobility-mass spectrometry (IM-MS) is a powerful analytical tool for structural characterization. IM measurement provides collision cross section (CCS) values that facilitate analyte identification. While CCS values can be directly calculated from mobility measurements obtained using drift tube ion mobility spectrometry (DT-IMS), this method has limited mobility resolution due to the practical constraints on the length of the ion drift path. Consequently, DT-IMS cannot differentiate analytes with similar mobilities or resolve fine mobility features of individual ions. Cyclic IMS (cIMS) instruments leverage a cyclic path enabled by traveling wave ion mobility (TWIM) technology and offer increased mobility solution to address this challenge. While TWIM devices must first be calibrated to enable CCS measurements, current calibration strategies are primarily tailored for single-pass analyses. This preference is partly attributed to the challenges associated with multipass calibration methods, which require both calibrants and analytes to experience the same number of passes. Achieving this consistency can be complicated due to factors like peak splitting and diffusion, and may not be feasible for online IM-MS analyses. A recent report employed average ion velocities obtained from multiple measurements under different separation pathlengths as a path length-independent metric for CCS calibration. However, the ability to exploit this averaging approach is limited by observed variation in ion drift time/velocity in these measurements. In this study, we introduce a novel calibration strategy designed for multipass cIMS analyses, directly targeting the root cause for the path length- and mobility-dependent variations in ion drift time. With this method, we demonstrate that CCS values derived from multipass measurements closely align with those obtained from single-pass analyses, with an average deviation of 0.1%. We apply this method to characterize four isomeric trisaccharides. Our approach not only results in excellent agreement between our measured cIMSCCS values and the reported DTCCS values, with an average difference of only 0.5%, but also allows us to effectively identify subtle mobility characteristics of each compound and determine their respective CCS values. This level of detail and accuracy was previously unattainable using DT-IMS or single-pass cIMS measurements. We developed an algorithm for reconstructing arrival time distribution in cases where wrap-around has resulted in peak splitting. Collectively, the new calibration strategy and the reconstruction procedure maintain reproducibility and precision in CCS measurements while largely eliminating the need for meticulous selection of separation times. We expect that our method will empower researchers to harness the high mobility resolution offered by multipass cIMS analyses without compromising the accuracy of CCS measurement, making it appropriate for straightforward use across a wide range of applications.

Leveraging High-Resolution Ion Mobility-Mass Spectrometry for Cyclic Peptide Soft Spot Identification.

Cyclic peptides are an important class of molecules that gained significant attention in the field of drug discovery due to their unique pharmacological characteristics and enhanced proteolytic stability. Yet, gastrointestinal degradation remains a major hurdle in the discovery of orally bioavailable cyclic peptides. Soft spot identification (SSID) of the regions in the cyclic peptide sequence susceptible to amide hydrolysis by proteases is used in the discovery stage to guide medicinal chemistry design. SSID can be an arduous task, traditionally performed using liquid chromatography-tandem mass spectrometry (LC-MS/MS), often resulting in complex and time-consuming manual analysis, particularly when isomeric linear peptide metabolites chromatographically coelute. Here, we present an alternative orthogonal approach that entails a high-resolution ion mobility (HRIM) system based on Structures for Lossless Ion Manipulation (SLIM) technology interfaced with quadrupole time-of-flight (QTOF) mass spectrometry to address some of the challenges associated with SSID. Two strategies were used to resolve linear isomeric peptide metabolites: labeled and label-free, both utilizing the HRIM platform. The label-free strategy leverages negative polarity to ionize the isomers which achieves better separation of the gas phase ions in the ion mobility (IM) dimension as compared to positive polarity, which is a more conventional approach when studying proteins and peptides. The second approach uses an isotope-labeled dimethyl tag on the terminal amine group, acting as a "shift reagent" to influence the mobility of isomers in the positive mode. This method resulted in baseline separation for the isomers of interest and produced unique product ions in the fragmentation spectra for unambiguous soft spot identification. Both label-free and labeled strategies demonstrated the ability to solve the challenges associated with SSID for cyclic peptides.

CIUSuite 3: Next-Generation CCS Calibration and Automated Data Analysis Tools for Gas-Phase Protein Unfolding Data.

Ion mobility-mass spectrometry (IM-MS) has become a technology deployed across a wide range of structural biology applications despite the challenges in characterizing closely related protein structures. Collision-induced unfolding (CIU) has emerged as a valuable technique for distinguishing closely related, iso-cross-sectional protein and protein complex ions through their distinct unfolding pathways in the gas phase. With the speed and sensitivity of CIU analyses, there has been a rapid growth of CIU-based assays, especially regarding biomolecular targets that remain challenging to assess and characterize with other structural biology tools. With information-rich CIU data, many software tools have been developed to automate laborious data analysis. However, with the recent development of new IM-MS technologies, such as cyclic IM-MS, CIU continues to evolve, necessitating improved data analysis tools to keep pace with new technologies and facilitating the automation of various data processing tasks. Here, we present CIUSuite 3, a software package that contains updated algorithms that support various IM-MS platforms and supports the automation of various data analysis tasks such as peak detection, multidimensional classification, and collision cross section (CCS) calibration. CIUSuite 3 uses local maxima searches along with peak width and prominence filters to detect peaks to automate CIU data extraction. To support both the primary CIU (CIU1) and secondary CIU (CIU2) experiments enabled by cyclic IM-MS, two-dimensional data preprocessing is deployed, which allows multidimensional classification. Our data suggest that additional dimensions in classification improve the overall accuracy of class assignments. CIUSuite 3 also supports CCS calibration for both traveling wave and drift tube IM-MS, and we demonstrate the accuracy of a new single-field CCS calibration method designed for drift tube IM-MS leveraging calibrant CIU data. Overall, CIUSuite 3 is positioned to support current and next-generation IM-MS and CIU assay development deployed in an automated format.

Evaluating Solid Phase Adsorption Toxin Tracking (SPATT) for passive monitoring of per- and polyfluoroalkyl substances (PFAS) with Ion Mobility Spectrometry-Mass Spectrometry (IMS-MS)

Detection and monitoring of per- and polyfluoroalkyl substances (PFAS) in aquatic environments has become an increasingly higher priority of regulatory agencies as public concern for human intake of these chemicals continues to grow. While many methods utilize active sampling strategies (“grab samples”) for precise PFAS quantitation, here we evaluate the efficacy of low-cost passive sampling devices (Solid Phase Adsorption Toxin Tracking, or SPATTs) for spatial and temporal PFAS assessment of aquatic systems. For this study, passive samplers were initially deployed in North Carolina along the Cape Fear River during the summer and fall of 2016 and 2017. These were originally intended for the detection of microcystins and monitoring potentially harmful algal blooms, though this period also coincided with occurrences of PFAS discharge from a local fluorochemical manufacturer into the river. Additional samplers were then deployed in 2022 to evaluate changes in PFAS fingerprint and abundances. Assessment of PFAS showed legacy compounds were observed across almost all sampling sites over all 3 years (PFHxS, PFOS, PFHxA, etc.), while emerging replacement PFAS (e.g., Nafion byproducts) were predominantly localized downstream from the manufacturer. Furthermore, samplers deployed downstream from the manufacturer in 2022 noted sharp decreases in observed signal for replacement PFAS in comparison to samplers deployed in 2016 and 2017, indicating mitigation and remediation efforts in the area were able to reduce localized fluorochemical contamination.

Hyphenation of supercritical fluid chromatography and trapped ion mobility-mass spectrometry for quantitative lipidomics

BackgroundLipidomics studies require rapid separations with accurate and reliable quantification results to further elucidate the role of lipids in biological processes and their biological functions. Supercritical fluid chromatography (SFC), in particular, can provide this rapid and high-resolution separation. The combination with trapped ion mobility spectrometry (TIMS) has not yet been applied, although the post-ionization separation method in combination with liquid chromatography or imaging techniques has already proven itself in resolving isomeric and isobaric lipids and preventing false identifications. However, a multidimensional separation method should not only allow confident identification but also provide quantitative results to substantiate studies with absolute concentrations. ResultsA SFC method was developed and the hyphenation of SFC and TIMS was further explored towards the separation of different isobaric overlaps. Furthermore, lipid identification was performed using mass spectrometry (MS) and parallel accumulation serial fragmentation (PASEF) MS/MS experiments in addition to retention time and collision cross section (CCS). Quantification was further investigated with short TIMS ramps and performed based on the ion mobility signal of lipids, since TIMS increases the sensitivity by noise filtering. The final method was, as an exemplary study, applied to investigate the function of different ceramide synthases (CerS) in the nematode and model organism Caenorhabditis elegans (C. elegans). Loss of three known CerS hyl-1, hyl-2 and lagr-1 demonstrated different influences on and alterations in the sphingolipidome. SignificanceThis method describes for the first time the combination of SFC and TIMS-MS/MS, which enables a fast and sensitive quantification of lipids. The results of the application to C. elegans samples prove the functionality of the method and support research on the metabolism of sphingolipids in nematodes.

Unsymmetric Chiral Ligands for Large Metallo‐Macrocycles: Selectivity of Orientational Self‐Sorting

Nature uses various chiral and unsymmetric building blocks to form substantial and complex supramolecular assemblies. In contrary, majority of organic ligands used in metallosupramolecular chemistry are symmetric and achiral. Here we extend on the group of unsymmetric chiral bile acids used as a scaffold for organic bispyridyl ligands employing the chenodeoxycholic acid (CDCA), epimer of previously used ursodeoxycholic acid (UDCA). Ligands’ epimerism, flexibility, and bulkiness leads to large structural differences of coordination products upon reaction with Pd(NO3)2. The UDCA‐bispyridyl ligand self‐assembles quantitatively into a single crown‐like Pd3L6 complex, whereas the CDCA‐ligand provides a mixture of coordination complexes of general formula PdnL2n, i.e., Pd2L4, Pd3L6, Pd4L8, Pd5L10, and even Pd6L12 containing impressive 120 chiral centers. The coordination products were studied by a combination of analytical methods, where the ion mobility‐mass spectrometry (IM‐MS) provided valuable details on their structure and allowed an effective separation of m/z 1461 to individual signals according to arrival time distribution, revealing four different ions of [Pd3L6(NO3)3]3+, [Pd4L8(NO3)4]4+, [Pd5L10(NO3)5]5+, and [Pd6L12(NO3)6]6+. The structures of all complexes were modelled using DFT calculations. Finally, challenges and conclusions in determination of specific structural identity of these unsymmetric species are discussed.

Native ESI-MS and Collision-Induced Unfolding (CIU) of the Complex between Bacterial Elongation Factor-Tu and the Antibiotic Enacyloxin IIa.

Collision-induced unfolding (CIU) of protein ions, monitored by ion mobility-mass spectrometry, can be used to assess the stability of their compact gas-phase fold and hence provide structural information. The bacterial elongation factor EF-Tu, a key protein for mRNA translation in prokaryotes and hence a promising antibiotic target, has been studied by CIU. The major [M + 12H]12+ ion of EF-Tu unfolded in collision with Ar atoms between 40 and 50 V, corresponding to an Elab energy of 480-500 eV. Binding of the cofactor analogue GDPNP and the antibiotic enacyloxin IIa stabilized the compact fold of EF-Tu, although dissociation of the latter from the complex diminished its stabilizing effect at higher collision energies. Molecular dynamics simulations of the [M + 12H]12+ EF-Tu ion showed similar qualitative behavior to the experimental results.

Carboxylated pillar[5]arene cavity accommodates organic cations in the host-guest complexes

The electron-rich cavity of carboxylated pillar[5]arene efficiently encapsulates cationic guest molecules within supramolecular assemblies. In this study we provide evidence of the host-guest complexation between carboxylated pillar[5]arene and benzamidine, a competitive inhibitor of serine proteases. We show the unexpected inclusion of the polar amidinium functionality within the central cavity of the macrocyclic host sustained by N+-H···π and cation···π interactions. In addition to the in-cavity complexation, simultaneous external binding of bipyridinium guests with varying stoichiometries has been observed. The interesting aspect is the inclusion of a hydrogen bonded pair of 1,2-bis(4-pyridyl)ethylene guests into pillar[5]arene cavity. Additional insights into inclusion characteristics are provided by NMR and ion mobility mass spectrometry (IM-MS) studies, offering information for the solution/gas phase and enabling a comprehensive description of the complexation process across different phases. This work expands the portfolio of cationic guest molecules capable of being encapsulated by carboxylated pillar[5]arene, showing new promises and directions for their inclusion and self-assembly behavior.

Exploring the Conformational Landscape of Poly(l-lysine) Dendrimers Using Ion Mobility Mass Spectrometry.

Ion mobility mass spectrometry (IM-MS) measures the mass, size, and shape of ions in the same experiment, and structural information is provided via collision cross-section (CCS) values. The majority of commercially available IM-MS instrumentation relies on the use of CCS calibrants, and here, we present data from a family of poly(l-lysine) dendrimers and explore their suitability for this purpose. In order to test these compounds, we employed three different IM-MS platforms (Agilent 6560 IM-QToF, Waters Synapt G2, and a home-built variable temperature drift tube IM-MS) and used them to investigate six different generations of dendrimers in two buffer gases (helium and nitrogen). Each molecule gives a highly discrete CCS distribution suggestive of single conformers for each m/z value. The DTCCSN2 values of this series of molecules (molecular weight: 330-16,214 Da) range from 182 to 2941 Å2, which spans the CCS range that would be found by many synthetic molecules including supramolecular compounds and many biopolymers. The CCS values for each charge state were highly reproducible in day-to-day analysis on each instrument, although we found small variations in the absolute CCS values between instruments. The rigidity of each dendrimer was probed using collisionally activated and high-temperature IM-MS experiments, where no evidence for a significant CCS change ensued. Taken together, this data indicates that these polymers are candidates for CCS calibration and could also help to reconcile differences found in CCS measurements on different instrument geometries.

Recent additions and access to a multidimensional lipidomic database containing liquid chromatography, ion mobility spectrometry, and tandem mass spectrometry information.

The importance of lipids in biology continues to grow with their recent linkages to more diseases and conditions, microbiome fluctuations, and environmental exposures. These associations have motivated researchers to evaluate lipidomic changes in numerous matrices and studies. Lipidomic analyses, however, present numerous challenges as lipid species have broad chemistries that require different extraction methods and instrumental analyses to evaluate and separate their many isomers and isobars. Increasing knowledge about different lipid characteristics is therefore crucial for improving their separation and identification. Here, we present a multidimensional database for lipids analyzed on a platform combining reversed-phase liquid chromatography, drift tube ion mobility spectrometry, collision-induced dissociation, and mass spectrometry (RPLC-DTIMS-CID-MS). This platform and the different separation characteristics it provides enables more confident lipid annotations when compared to traditional tandem mass spectrometry platforms, especially when analyzing highly isomeric molecules such as lipids. This database expands on our previous publication containing only human plasma and bronchoalveolar lavage fluid lipids and provides experimental RPLC retention times, IMS collision cross section (CCS) values, and m/z information for 877 unique lipids from additional biofluids and tissues. Specifically, the database contains 1504 precursor [M + H]+, [M + NH4]+, [M + Na]+, [M-H]-, [M-2H]2-, [M + HCOO]-, and [M + CH3COO]- ion species and their associated CID fragments which are commonly targeted in clinical and environmental studies, in addition to being present in the chloroform layer of Folch extractions. Furthermore, this multidimensional RPLC-DTIMS-CID-MS database spans 5 lipid categories (fatty acids, sterols, sphingolipids, glycerolipids, and glycerophospholipids) and 24 lipid classes. We have also created a webpage (tarheels.live/bakerlab/databases/) to enhance the accessibility of this resource which will be populated regularly with new lipids as we identify additional species and integrate novel standards.

Study of the Conformational Dynamics of Prolyl Oligopeptidase by Mass Spectrometry: Lessons Learned.

Ion mobility mass spectrometry (IM-MS) can be used to analyze native proteins according to their size and shape. By sampling individual molecules, it allows us to study mixtures of conformations, as long as they have different collision cross sections and maintain their native conformation after dehydration and vaporization in the mass spectrometer. Even though conformational heterogeneity of prolyl oligopeptidase has been demonstrated in solution, it is not detectable in IM-MS. Factors that affect the conformation in solution, binding of an active site ligand, the stabilizing Ser554Ala mutation, and acidification do not qualitatively affect the collision-induced unfolding pattern. However, measuring the protection of accessible cysteines upon ligand binding provides a principle for the development of MS-based ligand screening methods.

Four-dimensional lipidomics profiling in X-linked adrenoleukodystrophy using trapped ion mobility mass spectrometry

Lipids play pivotal roles in an extensive range of metabolic and physiological processes. In recent years, the convergence of trapped ion mobility spectrometry and MS has enabled 4D-lipidomics, a highly promising technology for comprehensive lipid analysis. 4D-lipidomics assesses lipid annotations across four distinct dimensions—retention time, collisional cross section, m/z (mass-to-charge ratio), and MS/MS spectra—providing a heightened level of confidence in lipid annotation. These advantages prove particularly valuable when investigating complex disorders involving lipid metabolism, such as adrenoleukodystrophy (ALD). ALD is characterized by the accumulation of very-long-chain fatty acids (VLCFAs) due to pathogenic variants in the ABCD1 gene. A comprehensive 4D-lipidomics strategy of ALD fibroblasts demonstrated significant elevations of various lipids from multiple classes. This indicates that the changes observed in ALD are not confined to a single lipid class and likely impacts a broad spectrum of lipid-mediated physiological processes. Our findings highlight the incorporation of mainly saturated and monounsaturated VLCFA variants into a range of lipid classes, encompassing phosphatidylcholines, triacylglycerols, and cholesterol esters. These include ultra-long-chain fatty acids with a length of up to thirty carbon atoms. Lipid species containing C26:0 and C26:1 were the most frequently detected VLCFA lipids in our study. Furthermore, we report a panel of 121 new candidate biomarkers in fibroblasts, exhibiting significant differentiation between controls and individuals with ALD. In summary, this study demonstrates the capabilities of a 4D-lipid profiling workflow in unraveling novel insights into the intricate lipid modifications associated with metabolic disorders like ALD.

Ion Mobility Mass Spectrometry-Based Disaccharide Analysis of Glycosaminoglycans.

Glycosaminoglycans (GAGs) are linear and acidic polysaccharides. They are ubiquitous molecules, which are involved in a wide range of biological processes. Despite being structurally simple at first glance, with a repeating backbone of alternating hexuronic acid and hexosamine dimers, GAGs display a highly complex structure, which predominantly results from their heterogeneous sulfation patterns. The commonly applied method for compositional analysis of all GAGs is "disaccharide analysis." In this process, GAGs are enzymatically depolymerized into disaccharides, derivatized with a fluorescent label, and then analysed through liquid chromatography. The limiting factor in the high throughput analysis of GAG disaccharides is the time-consuming liquid chromatography. To address this limitation, we here utilized trapped ion mobility-mass spectrometry (TIM-MS) for the separation of isomeric GAG disaccharides, which reduces the measurement time from hours to a few minutes. A full set of disaccharides comprises twelve structures, with eight possessing isomers. Most disaccharides cannot be differentiated by TIM-MS in underivatized form. Therefore, we developed chemical modifications to reduce sample complexity and enhance differentiability. Quantification is performed using stable isotope labelled standards, which are easily available due to the nature of the performed modifications.

Improvement of tumor neoantigen detection by high field asymmetric waveform ion mobility mass spectrometry.

Cancer neoantigens have been shown to elicit cancer-specific T-cell responses and have garnered much attention for their roles in both spontaneous and therapeutically induced antitumor responses. Mass spectrometry (MS) profiling of tumor immunopeptidomes has been used, in part, to identify MHC-bound mutant neoantigen ligands. However, under standard conditions, MS-based detection of such rare but clinically relevant neoantigens is relatively insensitive, requiring 300 million cells or more. Here, to quantitatively define the minimum detectable amounts of therapeutically relevant MHC-I and MHC-II neoantigen peptides, we analyzed different dilutions of immunopeptidomes isolated from the well-characterized T3 mouse methylcholanthrene (MCA)-induced cell line by MS. Using either data-dependent acquisition (DDA) or parallel reaction monitoring (PRM), we established the minimum amount of material required to detect the major T3 neoantigens in the presence or absence of high field asymmetric waveform ion mobility spectrometry (FAIMS). This analysis yielded a 14-fold enhancement of sensitivity in detecting the major T3 MHC-I neoantigen (mLama4) with FAIMS-PRM compared with PRM without FAIMS, allowing ex-vivo detection of this neoantigen from an individual 100 mg T3 tumor. These findings were then extended to two other independent MCA-sarcoma lines (1956 and F244). This study demonstrates that FAIMS substantially increases the sensitivity of MS-based characterization of validated neoantigens from tumors.

Time-Resolved Ion Mobility Mass Spectrometry to Solve Conformational Changes in a Cryptochrome.

Many biological mechanisms rely on the precise control of conformational changes in proteins. Understanding such dynamic processes requires methods for determining structures and their temporal evolution. In this study, we introduce a novel approach to time-resolved ion mobility mass spectrometry. We validated the method on a simple photoreceptor model and applied it to a more complex system, the animal-like cryptochrome from Chlamydomonas reinhardtii (CraCRY), to determine the role of specific amino acids affecting the conformational dynamics as reaction to blue light activation. In our setup, using a high-power LED mounted in the source region of an ion mobility mass spectrometer, we allow a time-resolved evaluation of mass and ion mobility spectra. Cryptochromes like CraCRY are a widespread type of blue light photoreceptors and mediate various light-triggered biological functions upon excitation of their inbuilt flavin chromophore. Another hallmark of cryptochromes is their flexible carboxy-terminal extension (CTE), whose structure and function as well as the details of its interaction with the photolyase homology region are not yet fully understood and differ among different cryptochromes types. Here, we addressed the highly conserved C-terminal domain of CraCRY, to study the effects of single mutations on the structural transition of the C-terminal helix α22 and the attached CTE upon lit-state formation. We show that D321, the putative proton acceptor of the terminal proton-coupled electron transfer event from Y373, is essential for triggering the large-scale conformational changes of helix α22 and the CTE in the lit state, while D323 influences the timing.

Anionic Conjugate Addition Oligomerization of Carbon Dioxide/Butadiene Derived Lactones.

Nucleophilic and non-nucleophilic bases have been employed in anionic oligomerization of unsaturated δ-valerolactone (3-ethylidene-6-vinyltetrahydro-2H-pyran-2-one) (1). Compared to the seminal findings with 1,5,7-triazabicyclo[4.4.0]dec-5-ene (TBD), the unsaturated lactone reacts with guanidines, disilazanes, and phosphazenes both in bulk and in solution with higher productivities and activities, reaching full conversion with turnover frequencies up to 382 h-1. Additionally, reactions using phosphazenes and NaHMDS were active at 1 mol % catalyst loadings both in solvent and in bulk monomer at room temperature. Characterization of the reaction products by 1H, 13C, FTIR, MALDI-MS, tandem mass spectrometry (MS/MS), and ion mobility mass spectrometry (IM-MS) revealed microstructural differences dependent on the nucleophilicity of the organocatalytic base and reaction conditions. The products from phosphazene-catalyzed reactions are consistent with selective vinylogous 1,4-conjugate addition, whereas both conjugate addition and ring-opening mechanisms are observed in TBD. DSC reveals that these microstructures can be tuned to have a Tg range between -18 and 80 °C, while SEC and MALDI-MS reveal that only low molar mass oligomers are formed (748-5949 g/mol). From these results, an approach for selectively favoring the vinylogous 1,4-conjugate addition pathway is obtained over ring-opening reactivity.

Utilization of bis-MPA Dendrimers for the Calibration of Ion Mobility Collision Cross Section Calculations.

Ion mobility-mass spectrometry (IM-MS) has become increasingly popular with the rapid expansion of available techniques and instrumentation. To enable accuracy, standardization, and repeatability of IM-MS measurements, the community requires reliable and well-defined reference materials for calibration and tuning of the equipment. To address this need, synthetic dendrimers of high chemical and structural purity were tested on three ion mobility platforms as potential calibrants. First, synthesized dendrimers were characterized by drift tube ion mobility (DTIMS), using an Agilent 6560 IM-qTOF-MS to assess their drift tube collision cross section (DTCCS) values. Then, assessment of obtained CCS values on trapped ion mobility (TIMS) and traveling wave ion mobility (TWIMS) ion mobility platforms were compared to those found by DTIMS. Across all three systems, dendrimers were found to have high potential for m/z and ion mobility calibration in the CCS range of 160-1700 Å2. To further validate their use as calibrants, drift tube calculated CCS values for dendrimers were utilized to calibrate calculations of CCS for known standards including Agilent Tuning mix, the CCS Major mix from Waters, and SPLASH LIPIDOMIX. Additionally, structures of sodiated dendrimers were computated along with theoretical CCS values which showed good agreement with the experimental CCS values. On the basis of the results presented, we recommend the use of dendrimers as alternatives and/or complementary compounds to commonly used calibrants for ion mobility platforms.